Neurochemical correlates of synaptically active amino acids

Life Sciences Vol. 15, pp. Printed in the U.S.A.

Pergamon Press

1045-1056

MINIREVIEW NEUROCHEMICAL CORRELATES OF SYNAPTICALLY ACTIVE AMINO ACIDS James P. Bennett, Jr., Arie H. Mulder and Solomon H. Snyder Departments of Pharmacology and Experimental Therapeutics and Psychiatry and the Behavioral Sciences The Johns Hopkins University School of Medicine Baltimore, Maryland 21205

Because acetylcholine, the first neurotransmitter studied in detail, is inactivated after its synaptic actions by enzymatic hydrolysis, for many years it was assumed that enzyme inactivation terminated the synaptic activities of all neurotransmitters.

However, extensive research with the bio-

genic amines, especially norepinephrine, has suggested that enzymatic inactivation of a neurotransmitter is an exception rather than the ruleo

The

universal means of inactivating neurotransmitters appears to be reuptake into the nerve ending which has released them as has been amply documented for norepinephrine, dopamine and serotonin (1,2,3).

Gamma-aminobutyric acid

(GABA) is the first amino acid for which strong evidence of a neurotransmitter role was obtained.

GABA also can be accumulated into nerve terminals

by a very specific high-affinity transport system (4,5). In addition to specific nerve terminal reuptake, recent evidence has suggested that glial cells may participate significantly in neurotransmitter inactivation.

High affinity uptakes for the biogenic amines and GABA have

been reported in glioma cells and bulk isolated glial fractions (which may be contaminated by nerve endings) [6,71 .

Since glial cell processes are known

to ensheath the synapse, glial transport of neurotransmitters could help regulate synaptic transmitter concentration and activity (8).

A variety of

neurochemical and neurophysiologic evidence has suggested that other amino acids, such as glutamic and aspartic acids and glycine might be neurotrans1045

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Neurochemically Active Amino Acids

mitters in the mammalian central nervous system If these amino acids are neurotransmitters,

Vol.

15, No. 6

(6,9,10,ii). identifying specific nerve

terminal uptake systems for them might provide evidence in support of their transmitter functions and might also afford a means of labeling the amino acid specific neurons.

Unfortunately,

unlike GABA,

amino acids such as

aspartic and glutamic acids and glycine subserve many general metabolic functions besides any neurotransmitter

roles.

It is well-known that these

as well as all other amino acids can be accumulated by virtually all animal cells via transport systems which ensure that the cells have adequate amino acid stores for incorporation into protein and intermediary metabolism. generalized amino acid transport systems have relatively low affinity

These (about

imM) [12] in contrast to the higher affinity of uptake systems for GABA and biogenic amines, whose affinity constants are about 0.i-i0 ~M (1-5). Accordingly,

we were pleased when it was possible in our (13) and other

(14-16) laboratories to identify high affinity uptake systems into synaptosomes (pinched-off nerve terminals)

of the mammalian nervous system for

glutamic and aspartic acids and glycine.

Numerous other amino acids which

are not thought to be neurotransmitters did not appear to possess such uptake systems.

The high affinity uptake system

for glycine could be demon-

strated with synaptosomal preparations from spinal cord and brain stem but not from the cerebral cortex, which accords nicely with the neurophysiologic evidence that glycine is a major inhibitory transmitter in the spinal cord and brain stem but not in the cerebral cortex.

Moreover,

it was possible to

show that these high affinity transport systems labeled unique populations of glycine,

aspartic and glutamic acid accumulating synaptosomes

(17-19).

The types of experiments which demonstrated unique populations of glycine, glutamic and aspartic acid accumulating synaptosomes involved incubating central nervous tissue under "high affinity" conditions with low amino acid concentration and centrifuging homogenates on continuous sucrose gradients capable of resolving various populations of synaptosomes

(20).

This accords

Vol. 15, No. 6

Neurochemically Active Amino Acids

1047

with the notion that these amino acids are transmitters in specific neuronal populations which possess physical properties differing from those of other neurons and which are labeled selectively by the high affinity uptake systems. Thus an abundance of evidence indicates that high affinity uptake systems for glycine and glutamic and aspartic acids label unique populations of nerve terminals in which these substances may serve a neurotransmitter role. Our initial experiments on amino acid uptake employed relatively crude homogenate systems which can be prepared more rapidly than "purified" synaptosomes.

Because these preparations cannot be "washed" they contain enough

endogenous concentrations of the amino acids that one cannot reliably study the uptake of lower than i0 ~M concentration of radioactive amino acid. serotonin precursor,

L-tryptophan,

tosomes by a high-affinity system

The

apparently is accumulated into rat synap(21,22),

and using purified synaptosomes

others reported high affinity accumulation of leucine into the cerebral cortex

(23).

Accordingly,

we reexamined the accumulation of numerous amino

acids by purified preparations of synaptosomes, between 1 and 10 ~M (Fig. i).

focusing on concentrations

For all amino acids studied in the cerebral

cortex, with the exception of glycine, we can demonstrate high affinity synaptosomal uptake.

To determine why high affinity uptake could not be

demonstrated for nontransmitter amino acids in earlier studies with cruder preparations,

we compared the kinetic data obtained under the two experi-

mental conditions.

The high affinity uptake systems for glycine,

glutamic

and aspartic acids are so avid that they are readily demonstrated in kinetic analysis using i0 ~M as the lowest amino acid concentration in incubations. However,

for the other amino acids the high affinity uptake system is less

prominent in relation to the low affinity transport and hence can only be demonstrated when amino acid concentration in the medium is reduced to the range 1-10 ~M.

Neurochemically Active Amino Acids

1048

Vol. 15, NO. 6

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FIG 1 Single-reciprocal (Eadie-Hofstee) plots of the accumulation of low concentrations (0.i-i0 ILM) of [3H~amino acids by purified nerve ending particle (synaptosome) fractions from adult rat cerebral cortex. Suspensions were incubated 4 min at 37°C and accumulated radioactivity assayed as previous!y described (37).

Sodium Dependence of Amino Acid Uptake.

Clearly the existence of the high

affinity synaptosomal uptake system for an amino acid of itself is not sufficient evidence for suggesting a neurotransmitter role for the compound. Studies of the ionic requirements for transport provide other criteria. synaptic uptake systems for norepinephrine,

serotonin,

manifest an absolute dependence on sodium (24-27). enate systems or purified synaptosomes

dopamine and GABA

Using either crude homog-

(Table I), we find that dependence on

sodium occurs only with a few amino acids, candidates.

The

specifically the neurotransmitter

The uptakes of low concentrations of glutamic and aspartic acids

are inhibited more than 95% by complete deletion of sodium both in cerebral cortex and spinal cord, while the uptakes of serine, tyrosine, leucine,

arginine,

alanine,

tryptophan,

and glycine are either reduced very little or

Vol.

1049

Neurochemically Active Amino Acids

15, No. 6

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Vol.

not at all by omission of sodium in the incubation medium.

15, No. 6

Glycine uptake

is highly sensitive to sodium deletion only in the spinal cord and not in the cerebral cortex,

again confirming the specificity of the high affinity

glycine uptake system for the spinal cord and brain stem. proline,

Strikingly,

not hitherto suspected as a neurotransmitter candidate,

up in a markedly sodium-dependent

fashion.

is taken

In the cerebral cortex proline

uptake is reduced 95% by the omission of sodium in the incubation medium. Selective Depolarization Induced Release of Amino Acid Neurotransmitter Candidates.

The existence of a sodium-dependent high affinity synaptosomal

uptake system for an amino acid seems to correlate with the possibility that such a compound has a neurotransmitter

function in the central nervous system.

Despite the heuristic value of high affinity uptake, criterion for identifying neurotransmitters.

it is an unorthodox

It certainly cannot be applied

rigidly or else one would have to conclude that acetylcholine does not qualifiy for neurotransmitter

status.

A more conventional criterion of neuro-

transmitters is that they be released when nerve terminals are depolarized. Numerous studies have been performed evaluating amino acid release from brain slices.

Several of these,

employing electrical depolarization,

strated release of almost all amino acids neurotransmitters specific.

(28).

have demon-

Either all amino acids are

or else the depolarizing stimuli are not sufficiently

In our own studies employing

electrical stimulation of slices

of cerebral cortex or spinal cord, we did find that almost all amino acids could be released from superfused tissue slices lished observations). was highly selective

(Mulder and Snyder,

unpub-

By contrast we found that potassium depolarization (29).

Fifteen mM potassium produces a significant

efflux of radiolabeled glycine from spinal cord slices with maximal effects at 42 mM.

By contrast very little potassium-induced

occurs from the cerebral cortex

(Table 2).

release of glycine

Unlike glycine,

[3H]glutamic

acid is released equally well in spinal cord and cerebral cortex but appears to be somewhat less sensitive to potassium than glycine in the spinal cord,

Neurochemically

Vol. 15, No. 6

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Active Amino Acids

TABLE 2 DEPOLARIZATION-INDUCED

RELEASE OF VARIOUS AMINO ACIDS FROM SLICES OF CENTRAL

NERVOUS SYSTEM TISSUE AFTER UPTAKE IN VITRO Cerebral Cortex

Acidic Amino Acids L-[14C]aspartic

acid

L-[14C] or [3H3glutamic acid

Spinal Cord

196 ± 29

(6)

139 ± 25

(5)

128 ± 19

(8)

115 ± 23

(7)

Basic Amino Acids L-[14C]arginine

-9 -+

L-[3H]histidine

(2)

-12

L-[14C]lysine

5

(2)

n.d.

4 +

2

(3)

9 +

3

[14C3 or [3H~glycine L-[14C3

7 +

5

(3)

(4)

16 -+ 8

(4)

36 -+ 2

(6)

201 -+ 20

(8)

or [3H]leucine

-7 -+ 6

(6)

-3 +

4

(4)

L-[14C]

or [3H]proline

74 +

9

(7)

32 +

5

(5)

L-[14C]

or [3H3serine

8 ±

4

(4)

29 -+ 4

(7)

209 +

9

(4)

74 ± i0

(6)

1 -+ 1

(6)

6 +

L-[14C3phenylalanine

6 -+ 3

(5)

n.d.

L-[14C] or [3H3tyrosine

5 +

(3)

Neutral Amino Acids L-[14C3alanine

[3H3~-aminobutyric

acid

[3H3~-aminoisobutyric

acid

4

(4)

0 +- 6

(5)

Aromatic Amino Acids

Slices were incubated

3

for 15 min with radiolabeled

~M and subsequently

trations

of 0.2-10

medium.

The major part of the data in this table were obtained from double-

label experiments.

Depolarization-induced

as the peak percentage efflux.

release by 40 mM K + is expressed spontaneous

for the number of experiments

Adapted from Mulder and Snyder

n.d.: not determined

with Krebs-Ringer-Tris

increase over immediate pre-stimulation

Values are means + S.E.M.

parentheses

superfused

amino acids at concen-

(29).

given in

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Neurochemically Active Amino Acids

Vol. 15, No. 6

since 42 mM potassium is required to induce glutamic acid efflux significantly greater than the basal level.

Perfusing with 42 mM potassium does not

release a variety of other tritiated amino acids from both cerebral cortex and spinal cord slices (Table 2).

We also found that the potassium stimu-

lated efflux of neurotransmitter candidate amino acids is dependent on calcium ions.

W h e n we removed calcium ions from the superfusion fluid we could

no longer stimulate the efflux of glutamic acid and glycine with potassium. This finding suggests that

potassium-stimulated effluxes are physiolog-

ically significant and consistent with the observed Ca-dependent release of other neurotransmitter candidates. One practical p r o b l e m with all in vitro release studies concerns the source or "pool" of the released material.

In particular, how do we k n o w

that the substance being released is coming from nerve terminals and not other cellular elements?

Certainly the selectivity we observe is consistent

with known synaptic activity or inactivity for the amino acids tested, but it is not enough by itself.

For the amino acids, we are fortunate because the

neurotransmitter candidates are accumulated into purified nerve ending preparations (synaptosomes) only in the presence of sodium ion (Table i).

When

we allow spinal cord slices to accumulate labeled glycine in the absence of sodium, the total amount of radioactivity accumulated is, as expected, much less than if sodium had been present.

However, enough radioactivity accumu-

lated for us to determine that 42 mM potassium can stimulate efflux over basal level.

Interestingly, for spinal cord slices labeled with glycine in

the absence of sodium potassium superfusion does not cause any increased efflux of glycine. Similarly [3H]GABA, [3H]glutamic acid, [3H]aspartic acid and [3H]proline can only be released from central nervous tissue slices if the slices have been labeled with amino acid in the presence of sodium (29). A similar dependence on sodium was observed previously in studies of unique populations of amino acid accumulating synaptosomes (19).

When incubated in

the presence of sodium and centrifuged on sucrose gradients by "incomplete

Vol. 15, No. 6

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Neurochemically Active Amino Acids

equilibrium sedimentation", [3H]glutamic and aspartic acids in cerebral cortex and [3H]glycine in spinal cord are localized to unique populations of synaptosomes which can be separated from the general population of synaptosomes which accumulate most other radiolabeled amino acids.

Synaptosomes

labeled with the acidic amino acids or glycine in the absence of sodium cannot be differentiated from the general population of synaptosomes (19). CONCLUSIONS The use of radiolabeled amino acids provides a powerful tool in evaluating possible transmitter characteristics of certain amino acids.

For GABA, glu-

tamic and aspartic acids and glycine, the neurophysiologic evidence suggesting t h e m as transmitters came first.

With the neurophysiologic evidence

in hand, we were in a p o s i t i o n to seek neurochemical correlates..

Having

identified unique neurochemical properties of amino acid neurotransmitter candidates, we are in a position to propose substances as possible transmitters on the basis of unique features of their biochemical disposition in the central nervous system.

Proline offers such an example.

It is accumu-

lated into cerebral cortical synaptosomes by a sodium-dependent high affinity transport system and is released in a selective fashion from brain slices by potassium depolarization.

We have not determlned whether unique

populations of proline accumulating synaptosomes can be separated from the general population of synaptosomes. We now suggest the following as valuable but not necessary neurochemical criteria for amino acids and possibly other neurotransmitter candidates in the brain.

In other words, the following criteria, if met, would greatly

favor a compound's candidacy as a neurotransmitter.

However, a neurotrans-

mitter might exist, such as acetylcholine, which does not satisfy the criteria; i.

High affinity sodium-dependent uptake into nerve terminals.

2.

Potassium-induced release from brain slices employing experimental

conditions under which other transmitters can be shown to be released selec-

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Neurochemically

Active Amino Acids

Vol.

15, No. 6

tively. 3.

Identification

of a unique population

of synaptosomes

accumulating

the compound in question. These criteria

are all pre-synaptic

ones.

the most crucial criteria are postsynaptic the actions of the natural transmitter

As suggested by Werman

ones.

The candidate must mimic

of the neuronal pathway

Until recently studies at the level of the postsynaptic quired neurophysiologic of the nicotinic eel or Torpedo cord

techniques.

cholinergic

(30),

in question.

receptor have re-

The recent biochemical

identification

receptor in electric organs of the electric

(31), the glycine receptor in mammalian brain stem and spinal

(32) and the muscarinic

cholinergic

provide powerful chemical probes

receptor in mammalian

brain

for identifying post-synaptic

(33-36)

neurotrans-

mitter actions in the brain. Supported by USPHS grant MH-18501, NIMH Research Scientist Development Award MH-33128 to S.H.S., a fellowship of the Netherlands Organization for Pure Research (ZWO) to A.H.M. and a grant of the John A Hartford Foundation.

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